Project Summary Ischemic heart diseases are the leading cause of morbidity and mortality. The underlying problems of these diseases are loss or dysfunction of blood vessels and insufficient new vessel formation. While cell therapy has emerged as a promising option to promote blood vessel growth, no therapy is yet clinically available and there is much room for improvement. The potential of bone marrow (BM)-derived cells turned out to be minimal9, and embryonic or induced pluripotent stem cell (ESC/iPSC)-derived ECs are difficult to maintain, costly to produce, and may cause side effects. To avoid these problems, a new approach, called direct reprogramming or direct conversion has been developed, in which somatic cells are converted into other lineage cells by overexpression of lineage- or cell-type specific transcription factors (TFs). This approach allows simpler and safer target cell generation and has the potential for more convenient clinical translation. We have attempted this direct reprogramming toward ECs using combinations of seven endothelial-related TFs and demonstrated for the first time that ETV2 alone is sufficient to convert human fibroblasts into ECs. However, since we used a lentiviral vector like others, these rECs have restrictions in clinical applicability. The direct EC reprogramming approach for therapy has two options: cell therapy or direct in vivo reprogramming. For clinical application, both require a safer delivery vector to minimize the possibility of genomic integration. Thus, we developed an adenoviral-ETV2 (Ad-ETV2) vector. Another important obstacle for cell therapy is short- term survival of the transplanted cells. To overcome this problem, we have investigated the potential of biomaterial for prolongation of the cell survival and therapeutic effects. We recently showed that peptide amphiphile (PA) nanomatrix gel is very effective, extending survival of human iPSC-derived ECs longer than 10 months and inducing continuous vessel formation in vivo. In this study, first, we will first develop cell-based therapy. We will generate clinically compatible rECs from human fibroblasts and generate an optimal PA construct combining these rECs with various types of PA nanomatrix gel including a newly developed nitric oxide (NO)-releasing PA. We will then determine the vascularization and therapeutic effects of the selected rEC-PA nanomatrix constructs on rodent ischemic heart disease models. Second, we will investigate whether direct delivery of Ad-ETV2 into cardiac ischemia models can directly reprogram fibroblasts into functional endothelial cells and induce vascularization in vivo. We will use genetically modified mice to track the fate of fibroblasts toward ECs in vivo. The goal of this project is to develop clinically applicable cardiac revascularization strategy using a novel direct reprogramming approach combined with tissue engineering technologies. If successful, this study will provide next-generation platforms for broad areas of research and therapy for ischemic heart diseases.